Project Summary Microbial natural products are known to be rich resources for the search of drug candidates. Advances in next generation sequencing have exponentially increased the metagenomic data available from actinomycetes, which are known to be prolific producers of bioactive secondary metabolites. However, much of natural product chemical diversity has not been explored using traditional, cultivation-dependent strategies. Furthermore, a significant fraction of gene clusters from cultivated microbial sources remain silent under standard fermentation conditions. In theory, heterologous expression of natural product gene clusters can provide access to chemical diversity from both uncultivated organisms and cryptic pathways. However, current cell-based heterologous expression approach remains to be the biggest bottleneck for genomic mining as often times it can be very time consuming and expensive, and usually limited to a small number of standard hosts. In Phase I, will this bottleneck by applying our cell-free expression technology to heterologously express cryptic actinomycete gene clusters in vitro. Cryptic biosynthetic gene clusters of natural products with therapeutic value can be characterized in a high-throughput and cost-effective fashion. We propose to achieve this objective through three specific aims. Aim 1. Platform development of diverse actinomycetes cell-free systems. We anticipate that cell-free systems from multiple actinomycete strains will improve the probability of cryptic cluster heterologous expression. Aim 2. Identification of cryptic actinomycetes biosynthetic pathways of natural products. We will identify never before expressed cryptic actinomycetes biosynthetic pathways and filter based on expression potential in our cell-free expression platform. Aim 3. High throughput in vitro cell-free expression. We will run high-throughput (384+) experiments to then express and characterize 3 unique cryptic gene clusters. If successful, this valuable platform can be used to activate the pool of bioinformatically-available cryptic biosynthetic gene clusters for the discovery of new natural products from actinomycetes with high pharmacological value. Phase II efforts will focus on platform scale up and the identification and scaling of potential therapeutic candidates for downstream characterization.